KR101934781B1 - Medium for culturing plantlets in vitro of moringa plant - Google Patents

Abstract

The present invention relates to a growth medium for growing a differentiated Moringa plant tissue to a complete vegetable plant, comprising 30 g to 40 g / L of sucrose, 30 g / L of D-sorbitol, inositol and agar in an amount of 8 to 10 g / L. Thus, the plant tissue obtained from the stem, roots and leaves is differentiated from the plant cultured in the plant, And it can be cultivated in a large quantity simply because it directly utilizes the moringa tissues cultivated in the cabin, and because of its high growth rate, it can be formed by increasing the number of seedlings in the proliferation culture process for expanding the culture scale It is possible to grow the whole plants in a short period of 4 weeks, so that the seedlings can be supplied to the farmers in a large amount. Thus, even in the domestic climate environment in which the cultivation period is from May to October, And to be able to harvest the roots can get the import substitution effect of increasing farmers' income and Moringa.

Description

{Mining for culturing plantlets in vitro of moringa plant}

The present invention relates to a growth medium for Moringa's in-flight plant formation, and more particularly to a growth medium for growing a plant tissue that is differentiated from stem, root, and leaf in a Moringa plant into a full-fledged plant.

Moringa Oleifera is a perennial leguminous plant belonging to the Moringaceae family, which is distributed in tropical and subtropical climatic regions. It is a shrub ranging from 5 to 12 meters in length, It is native to the northwestern part of India.

Mouring contains more than 90 nutrients including 16 vitamins, 17 minerals, 20 essential amino acids, 15 fatty acids, and 3, 6, and 9 omega. Is the most abundant plant nutrient. It contains more than 90 nutrients needed for the human body and it is reported as "Miracle Tree".

It is known that Moringa has many effects such as prevention of cholesterol, anti-inflammation, improvement of eyesight, hypertension, immunity enhancement, antioxidation, anti-aging, tumor prevention and diabetes. In particular, Moringa leaf is known to have various effects such as protein, calcium, iron, Vitamin A and Vitamin E), Vitamin C and Vitamin E are very abundant, and it is possible to increase the natural defenses of the body by various active ingredients, provide nutrition to eyes and brain, promote metabolism, improve cell structure, improve wrinkles, normalize liver and kidney Its functional value, digestive function, antioxidant effect, immune system enhancement, anti-inflammatory action and blood sugar co-action.

These moring herbs have been cultivated in India, Vietnam, the United States, and the Philippines and have been processed and sold as oil, dried leaves and powder. Recently, however, Moringa has been cultivated in Japan and China. Accordingly, the utility of functional health supplements and functional cosmetics is expanding, and demand is also increasing.

As a result of the widespread nutritional overgrowth such as obesity, adult disease, gout, and metabolic syndrome which are common in developed countries due to the economic growth of Korea, the demand for health functional foods has been greatly increased due to increased interest in health, , There is an increasing effort to utilize the functional ingredients obtained from the existing plants as food materials, and the public is increasingly interested in the Moringa introduced through the media.

As a result, Moringa was introduced to Korea about 4 ~ 5 years ago. In Korea, imports, processing, and sales of foreign direct products and raw materials are mainly imported. Although the import amount of Moringa is rapidly increasing, , Which is estimated to be in the range of 50 ~ 60, is leading to cultivation in Cheorwon, the jurisdiction of the present applicant.

Moringa is a perennial plant that grows in 20 ~ 30 years. It grows well in the tropics but can not grow in the domestic cold winter season. Domestically, it can be cultivated from early May to late October. to be.

In Korea, it is necessary to harvest the desired leaves, stems, roots, etc. for about 5 months, so that it is not possible to collect the seeds because the cultivation period is short. However, since the interest of the farmers is increased, However, since most of the seeds currently distributed in Korea are supplied from Thailand, Vietnam, India, and the Philippines, and these imported seeds are used for edible and processing purposes, such as seeds, Is 10 to 15%, which is extremely low.

Therefore, it is very necessary to secure domestic production technology of tissue culture seedlings which can be used as a disease-free disease free from viruses, root growth is fast, initial growth rate is high,

The culture method that induces shoot formation and plant regeneration through callus (callus, or union tissue, wound tissue, oily tissue), which refers to an indefinite amorphous cell mass, is a method that utilizes the totipotency of a plant Refers to the ability of whole plants to regenerate from a single cell or part of plant tissue. All cells have this typical performance, but the results expressed by the degree of differentiation of the cell tissue, the harvesting site, the composition of the medium, the culture environment, etc. are significantly different, and the cultivation period is very short from May to October In order to supply a large amount of Moringa seedlings in accordance with the domestic situation, optimal Moringa planting technology for planting by callus cultivation is required.

It is an object of the present invention to provide a plant which is differentiated from any one selected from the stem, root and leaf of a Moringa plant cultivated in the cabin according to domestic circumstances in which mass production of the seedling of Moringa is inevitably carried out through tissue culture according to the domestic climate environment To provide a growth medium for the formation of Moringa inflorescence plants which are improved to grow the tissue into a full-fledged plant in a short period of time.

In order to achieve the above object, the growth medium for forming in-cabbage plants of Moringa according to the present invention comprises 30 g to 40 g / L of sucrose, 30 g / L of D-sorbitol, 0.1 g / L of inositol and 8 to 10 g / L of agar.

Preferably, the basal medium is MS (Murashige & Skoog) medium or WPM (Lloyd & McCown Woody Plant Basal Salt Medium) medium.

The basal medium may be a medium prepared by mixing MS (Murashige & Skoog) medium and WPM (Lloyd & McCown Woody Plant Basal Salt Medium) at a volume ratio of 1: 1.

The growth medium contained KNO3 2,850 mg / L, NH4NO3 2,470 mg / L, CaCl2H2O 660 mg / L, MgSO47H2O 550 mg / L, KH2PO4 255 mg / L, MnSO44H2O 33.1 mg / L, ZnSO47H2O 12.3 mg / L, H3BO3 9.1 mg / Inorganic salts of glycine 3 mg / L, myo-inositol 250 mg / L, NaClO2H2O 0.037 mg / L, NaCl2H2O 0.037 mg / L, FeSO47H2O 41.7 mg / L, Na2EDTA2H2O 55.9 mg / , 0.75 mg / L of nicotinic acid, 0.75 mg / L of pyridoxine HCl, 0.15 mg / L of thiamine HCl, 3% of sucrose and 3% of sorbitol.

The growth medium for planting Moringa according to the present invention is a plant tissue differentiated from a plant cultivated in stem, root and leaf, and is induced to grow into a milk plant in a short period of time. Because of its direct use of tissue, it can be cultivated in large quantities in a simple manner, and because of its high growth rate, it can be formed by increasing the number of endemic plants in the proliferative culture process, And it is possible to harvest the leaves, stalks and roots in the farmhouse even in the domestic climate environment where the cultivation period is short from May to October, Import substitution effect can be obtained.

1 is a photograph showing growth processes of differentiated plant tissues of Moringa in a growth medium according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to specific examples.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

Studies on the production techniques in the Moringa tissue culture medium carried out in the present invention have shown that the growth of the plant from the differentiated plant tissues of Moringa's stem, roots and leaves cultivated in-flight for the mass production of Moringa seedlings to full- In the culture medium.

That is, since the growth of plant tissues differentiated from the stem, root and leaf of Moringa differs depending on the kind and concentration of the additives including the growth regulator, when the tissue culture method is used, the planting rate of Moringa is increased, In order to mass-produce successful tissue culture seedlings, it is necessary to optimize the composition of the medium such as the type and concentration of the medium supplement including the growth regulator.

The growth medium according to the present invention is used for growth of plant-derived tissues differentiated from stems, roots and leaves of Moringa seedlings of a disease-free seedling. The above-mentioned Moringa seeds were selected by selecting a large and robust seed, immersed in 70% ethanol for 10 seconds as a pretreatment, washed 3 to 4 times with sterilized distilled water, immersed in 1% sodium hypochlorite solution for 15 to 20 minutes , And the cells are washed again with sterilized water three to four times. After removing the moisture of the seed using sterilized filter paper, the cells are cultured on an agar medium or a phytagel medium. In this preliminary step, Among the seedlings, those having good vitality are selected and differentiated and grown in the growth medium according to the present invention.

To this end, the growth medium for growing the Moringa seedlings according to the present invention comprises 30 to 40 g / L of sucrose, 30 g / L of D-sorbitol, 0.1 g / L of inositol, And 8 to 10 g / L of agar (Agar).

Tissue culture medium is an inorganic salt of tissue culture medium excluding oxygen, carbon and hydrogen, including inorganic salts, vitamins, growth regulators, carbon and energy sources, other organic substances and other additives (agar, Elements required for cell and tissue culture include trace elements such as nitrogen, phosphorus, calcium, calcium, magnesium and sulfur and trace elements required in relatively small amounts such as calcium, sodium, manganese, molybdenum, copper, zinc, boron, cobalt and iron do.

These elements play an important role in the development of not only plants but also cells and tissues. Particularly, P is a component of nucleic acid and is essential for cell division and growth. Ca is a constituent of cell wall and it binds to pectin in middle lamella, It plays an important role in cell division and growth. Iron (Fe) in trace elements plays a role as a cofactor to complement and assist in the function of enzymes that regulate various metabolic processes occurring in plants. It also plays an important role in catalyzing photosynthesis, such as the transfer of electrons through redox action, and is also a component of respiratory enzymes.

The basal medium used in the present invention can be prepared by using MS (Murashige & Skoog Medium, Basal Salt Mixture, NH4NO3 Free) or using another medium such as WPM (Lloyd & McCown Woody Plant Basal Salt Medium) It is possible. In addition, the MS medium and the WPM medium may be mixed at a weight ratio of 1: 1. The basal medium contained only the most basic substances necessary for plant cultivation. In addition, the pH of the basic medium was used in the range of 5.8 to 6.0.

The growth medium of the present invention was subjected to the following procedure for the growth of the differentiated milk plants of Moringa. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Examples>

Moringa's George culture medium composition

<1> Culture for moringa growth induction

L of Sucrose, 30 g / L of D-sorbitol, 0.1 g / L of inositol and 10 g / L of inositol were added to the basic medium to grow the differentiated plants in Moringa. ~ 10g / L were used as the growth medium and cultured for 40 days.

Each of the medium components was mixed and prepared, and 100 to 150 mL of the culture medium was dispensed in a culture bottle (for example, outer diameter: 81 mm, height: 132 mm), and sterilized at 121 캜 for 15 minutes. Ten plant tissues were streaked into the culture medium containing the medium, and cultured for about 40 days in a culture room maintained at 26 ± 1 ° C.

The components of the culture medium of the growth medium according to the present invention are shown in Table 1 below.

Growing medium composition of Moringa division Ingredients Content (mg / L) Inorganic salts
(Heavy element) KNO3 2,850 NH4NO3 2,470 CaCl22H2O   660 MgSO47H2O   550 KH2PO4   255 Inorganic salts
(Trace element) MnSO44H2O      33.1 ZnSO47H2O      12.3 H3BO3       9.1 KI       1.2 CuSO45H2O         0.037 Na2MoO42H2O        0.37 CoCL26H2O         0.037 FeSO47H2O      41.7 Na2EDTA2H2O      55.9 Organic substance glycine     3 myo-inositol   250 nicotinic acid        0.75 pyridoxineHCl        0.75 thiamine HCl        0.15 sucrose 3% sorbitol 3%

According to the present invention, it is possible to add 30 g to 40 g / L of sucrose, 30 g / L of D-sorbitol, 0.1 g / L of inositol and 8 to 10 g / L of Agar to the base medium Growth medium and 10 control plants to be cultivated in the culture medium containing the basic medium without sorbitol and inositol were added to the growth medium and cultured in a culture room maintained at 26 ± 1 ° C for about 40 days.

At this time, the growth state of the differentiated milk plants of Moringa at the time when 20 days and 40 days of cultivation had passed from the wounded state was confirmed by photograph as shown in FIG. In the photographs of each growth state in Fig. 1, the left photograph shows the growth state of the seedling plants in the growth medium in which sorbitol and inositol were added to the basic medium, and the right photograph shows growth of the seedling plants in the medium with only the basic medium as the control group The situation is shown in contrast.

As can be seen in the leftmost photograph in FIG. 1, there was no significant difference in the state of the differentiated milk plants in the growth medium and the control medium of the present invention. However, in the growth medium of the present invention at 20 days of culture, the number of leaves of the stemmed plant was higher than that of the control medium (more dark green in the photograph), and after 40 days of culture, Of the moringa seedlings were significantly larger and larger than those of the control seedlings of Moringa seedlings, and the number of leaves was also larger. Specifically, from the results shown in Table 2 below, the planting rate , There was a significant difference in leaf number and plant height.

Comparison of growth characteristics of cultured plant tissues differentiated into growth medium supplemented with 30 g / L of sorbitol (D-sorbitol) and 0.1 g / L of inositol in the basic medium and medium containing only basic medium division Plant
Formation rate (%) Number of shoots
(plantlet) ground game
(leaf) Plant height
(mm) Remarks Addition medium 72.5 ± 7.2 4.5 ± 4 72.5 ± 7.2 135.5 ± 13.5 4 parking Non-added medium 47.5 ± 4.7 2.8 ± 2 35.4 ± 3.5 67.2 ± 6.7 4 parking

In addition, in the growth medium of the present invention, the plant growth characteristics of plant tissues differentiated from the tissues of callus, axillary eye region (including the growth point), stem node (including the growth point) and root (including the growth point) Table 3 shows that the plant growth rate, number of shoots, number of leaves, and plant height were good in the growth medium of the present invention in any tissues.

Plant growth characteristics division Plant
Formation rate (%) Number of shoots
(plantlet) ground game
(leaf) Plant height
(mm) Remarks Callus 52.5 ± 5.2 3.7 ± 3 75.4 ± 7.5 94 ± 9.4 24 parking The ax side of the stem 65.5 ± 6.5 3.5 ± 3 78.5 ± 7.8 91.4 ± 9.1 8 parking Stem node 72.5 ± 7.2 4.5 ± 4 72.5 ± 7.2 135.5 ± 13.5 8 parking Root 50.5 ± 5.0 3.2 ± 3 72.5 ± 7.2 71.4 ± 7.1 8 parking

From this, it can be confirmed that the growth medium according to the present invention is very useful for growth of plant tissues of callus, axillary eye region, stem node region and root region of Moringa.

<2> Purification process for morphogenesis cultivation of Moringa

Tissue cultures cultured in the growth medium become independent complete plants only through an external purification process. During fertilization, it forms shoots, anterior and lateral roots, and continues nutrition growth. It is planted in a tray (27x53x12.5cm, 50gu) containing general peat moss for gardening, shielding 30 ~ 40% of sunlight and covering transparent vinyl in greenhouse keeping maximum 25 ℃ and minimum 18 ℃, . When new leaves were formed, they gradually increased the light intensity and lowered the humidity. Then, while checking the condition of the plants, the vinyl was gradually removed and cured by fully exposed to the environment of the outside air.

Although MS (Murashige & Skoog) is used as a primary medium in the above description of the embodiment, it is not limited thereto. For example, another medium such as WPM (Lloyd & McCown Woody Plant Basal Salt Medium) , There was no significant difference in the growth characteristics of the seedling plants when the MS medium was used for the growth characteristics of the medium in the medium in which the MS medium and WPM medium were mixed at a ratio of 1: 1 as the basic medium.

The growth medium for the formation of the Moringa herbaceous plant according to the present invention is a perennial soybean plant distributed in a subtropical region, which is a tropical plant which has not been attempted because the domestic climate is not suitable for cultivation through the tissue culture of the herbaceous plant. It can be used for mass production of Moringa seeds containing nutrients.

Claims (5)

In a growth medium for growing a differentiated moringa plant tissue into a full-fledged plant,
Sucrose 30g to 40g / L, Sorbitol (D-glucose), and Sorbitol were added to a medium containing MS (Murashige & Skoog) medium and WPM (Lloyd & McCown Woody Plant Basal Salt Medium) at a volume ratio of 1: sorbitol), inositol (0.1 g / L) and agar (8 to 10 g / L).
delete delete delete The method according to claim 1,
The growth medium contained KNO3 2,850 mg / L, NH4NO3 2,470 mg / L, CaCl2H2O 660 mg / L, MgSO47H2O 550 mg / L, KH2PO4 255 mg / L, MnSO44H2O 33.1 mg / L, ZnSO47H2O 12.3 mg / L, H3BO3 9.1 mg / Inorganic salts of glycine 3 mg / L, myo-inositol 250 mg / L, NaClO2H2O 0.037 mg / L, NaCl2H2O 0.037 mg / L, FeSO47H2O 41.7 mg / L, Na2EDTA2H2O 55.9 mg / , nicotinic acid 0.75 mg / L, pyridoxine HCl 0.75 mg / L, thiamine HCl 0.15 mg / L, sucrose 3% and sorbitol 3%. badge.

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